As the most common solid tumor in children, neuroblastoma accounts for 8-10% of all cancers in children (for review see Lee et al., 2003, Urol. Clin. N. Am. 30, 881-890). Annual incidence ranges from 10 to 15 per 100,000 infants, according to population based screening conducted in Canada, Germany and Japan. Neuroblastoma is a heterogeneous disease, with 40% diagnosed in children under 1 year of age who have a very good prognosis, and the rest in older children and young adults who have a poor prognosis despite advanced medical and surgical management. A common treatment for intermediate- and high-risk patients is chemotherapy followed by surgical resection. However, complete eradication of neuroblastoma cells is seldom achieved. Consequently, the majority of these patients undergo relapse, which is often resistant to conventional treatment and rapidly overwhelming. Thus, after induction of the apparent remission by the first-line therapy, new therapeutic strategies are needed to completely eradicate the small number of surviving cells, to prevent relapse (Lee et al., 2003, supra).
Brain development requires the co-ordinated and precise patterning of cell division, migration and differentiation of neuroblasts (Noctor et al., 2001, Nature 409, 714-720; Noctor et al., 2004, Nat. Neuroscience 7, 136-144). A key event in both these processes is the (re)organization and (de)stabilization of the cytoskeleton, which is comprised of microtubules and microtubule-associated proteins (MAPs). A carefully orchestrated interaction of microtubules with several MAPs is required before neuronal migration can occur (reviewed in Feng and Walsh, 2001, Nat. Rev. Neurosci. 2, 408-416). Although the factors involved in neuronal migration are well established, relatively little is known about the genes that control earlier processes, like mitosis and neuroblast proliferation. Such factors very likely involve dynamic regulation of the microtubular and cytoskeletal elements as well (Haydar et al., 2003, Proc. Natl. Acad. Sci. 100, 2890-2895; Kaltschmidt et al., 2000, Nat. Cell Biol. 2, 7-12; Knoblich, 2001, Nat. Rev. Mol. Cell Biol. 2, 11-20).
Recently, several genes involved in cytoskeleton reorganization have been identified that, when disrupted or mutated, cause neuronal migration disorders (reviewed in Feng and Walsh, 2001, supra). One of these genes is doublecortin (DCX) that encodes a 365 AA protein critical for migration of newborn cortical neurons (see WO99/27089). In the human and rodent genome, a related gene, called doublecortin-like kinase (DCLK), is present that has substantial sequence identity with the DCX gene. The human DCLK gene spans more than 250 kb and is subject to extensive alternative splicing, generating multiple transcripts encoding different proteins (Matsumoto et al., 1999, Genomics 56, 179-183). One of the main transcripts, DCLK-long, encodes a DCX domain fused to a kinase-like domain that has amino acid homology with members of the Ca++/Calmodulin dependent protein kinase (CaMK) family. Another transcript, DCLK-short, is mainly expressed in adult brain, lacks the DCX domain and encodes a kinase with CaMK-like properties (Engels et al., 1999, Brain Res. 835, 365-368; Engels et al., 2004, Brain Res. 120, 103-114; Omori et al., 1998, J. Hum. Genet. 43, 169-177; Vreugdenhil et al., 2001, Brain Res. Mol. Brain Res. 94, 67-74). Recent studies suggest important roles for the DCLK gene in calcium-dependent neuronal plasticity and neurodegeneration (Burgess and Reiner, 2001, J. Biol. Chem. 276, 36397-36403; Kruidering et al., 2001, J. Biol. Chem. 276, 38417-38425). DCLK-long is expressed during early development (Omori et al, 1998, supra) and like DCX, is capable of microtubule polymerization (Lin et al., 2000, J. Neurosci. 20, 9152-9161). However, the precise role of the DCLK gene in development of the nervous system is unknown.
Various alternative splice-variants of DCLK have been described and two of these have been found to be differentially expressed and to have different kinase activities (Burgess and Reiner 2002, J. Biol. Chem. 277, 17696-17705). The present inventors cloned and functionally characterized a novel splice variant of the DCLK gene, referred to as doublecortin-like (DCL) herein, and have shown that DCL is a cytoskeleton gene which is associated with mitotic spindles of dividing neuroblasts. In addition, the present inventors have devised novel methods for cancer therapy and diagnosis, especially for neuroblastoma therapy and diagnosis.
Recently, new approaches for treatment of neuroblastoma have been published, involving the use of antisense oligonucleotides targeting two different oncogenes (Pagnan et al., 2000, J. Natl. Cancer Inst. Vol 92, 253-261; Brignole et al. 2003, Cancer Lett. 197, 231-235; Burkhart et al., 2003, J. Natl. Cancer Inst. 95, 1394-1403). The first approach was directed against the c-Myb oncogene (Pagnan et al., 2000, supra). C-Myb gene expression has been reported in several solid tumors of different embryonic origins, including neuroblastoma, where it is linked to cell proliferation and differentiation. It was shown that a phosphorothioate oligodeoxy-nucleotide complementary to codons 2-9 of human c-Myb mRNA inhibited growth of neuroblastoma cells in vitro. Its inhibitory effect was greatest when it was delivered to the cells in sterically stabilized liposomes coated with a monoclonal antibody (mAb) specific for the neuroectoderma antigen disialoganglioside GD2 (Pagnan et al., 2000, supra). Although pharmaco-kinetic and biodistribution studies after intravenous injection of anti-GD2-targeted liposomes have been performed (Brignole et al., 2003, supra), the effect in an in vivo neuroblastoma model has not been shown so far. Potential toxic side-effects of a c-Myb antisense oligonucleotide should also be considered, since the c-Myb protein plays a fundamental role in the proliferation of normal cells and it has already been shown that a c-Myb antisense oligonucleotide inhibits normal human hematopoiesis in vitro (Gewirtz and Calabretta, 1988, Science 242, 1303-1306).
Another antisense approach was directed against the MYCN(N-myc) oncogene (Burkhart et al., 2003, supra). Amplification of the MYCN gene occurs in only 25 to 30% of neuroblastomas, but is associated with advanced-stage disease, rapid tumor progression and a survival rate of less than 15%. The effect of a phosphorothioate oligodeoxynucleotide complementary to the first five codons of human MYCN mRNA was tested in vivo in a murine model of neuroblastoma. It was shown that continuous delivery of the oligonucleotide for 6 weeks via a subcutaneously implanted microosmotic pump could decrease tumor incidence and tumor mass at the site of the implanted pump (Burkhart et al., 2003, supra). This approach is very local however, and a systemic effect of the oligonucleotide on metastases to distant organ sites remains to be established, in addition to potential toxic side effects on normal cells after systemic delivery. Also, the effect of the oligonucleotide on an already established tumor has not been shown.
The choice of the target gene is crucial for the development of an effective neuroblastoma therapy and diagnosis. As mentioned above, the present inventors have cloned a novel mRNA splice variant of the DCLK gene, encoding the novel DCL protein, and have functionally characterized this splice variant. It was surprisingly found that this splice variant is exclusively expressed in neuroblastomas, while not being detectable in the healthy tissue and cell lines tested. This finding was used to devise novel therapeutic and diagnostic methods.